Tumor gene expression in vitro and in vivo

我们的生长调控研究一直关注癌基因作为正常和肿瘤生长的正调控因子和肿瘤抑制基因作为负调控因子。目前主要项目集中在肿瘤抑制基因dcl1的分子生物学研究，包括调控dcl1的靶点及其调控的靶点。dcl1在多种肿瘤中经常失活，但其作用机制的许多方面仍不完全清楚。DLC1通过其Rho- gap活性负向调节Rho，并编码其他活性。一个主要的假设是，dcl1在癌症中经常失活，因为它编码一种重要的生物学多功能蛋白。为了支持这种可能性，我们之前已经确定DLC1与：1)张力蛋白相互作用，通过DLC1的n端区域，该区域之前没有发现任何功能；2) focal adhesion kinase （FAK）和talin，通过共享的8个氨基酸基序；3)通过DLC1 c端附近的star相关脂质转移（START）结构域表达的小洞蛋白-1 （CAV-1）。DLC1突变体分析表明，这些相互作用的结合位点都有助于DLC1的生长抑制功能，但突变体的结合减少并不影响DLC1的体内Rho-GAP活性。这些研究验证了DLC1是一种多功能蛋白的假设，其生物活性既取决于其Rho-GAP活性，也取决于其结合多种信号分子的能力。我们还评估了TCGA中12种癌症类型中DLC1和其他9种rho - gap点突变的存在。结果表明，DLC1是Rho-GAP突变最频繁的，在评估的五种肿瘤类型中，有5-8%的肿瘤发生DLC1错义突变。此外，在这五种肿瘤类型中的四种中，20-26%的肿瘤在10个rho - gap中至少有一个存在错义突变。对DLC1突变体的实验分析表明，在9个位于Rho-GAP结构域的突变体中，有7个突变体缺乏Rho-GAP活性、抑制细胞迁移和非锚定生长。dcl1的点突变分散在基因的编码序列中，这意味着重要的生物学功能存在于整个蛋白质中。这些结果表明，除了基因缺失和表观遗传改变外，dcl1的点突变是其在癌症中功能下降的重要机制。我们已经描述了dcl1的翻译后修饰，特别是磷酸化如何影响其活性。我们首先确定了CDK5，一种细胞质激酶，其生理作用促进分化，作为DLC1的主要激活因子，通过CDK5磷酸化DLC1中的4个丝氨酸发生。当这些位于dcl1 RHO-GAP结构域n端的丝氨酸未被磷酸化时，n端与RHO-GAP结构域结合，使dcl1处于封闭的非活性构象。当丝氨酸被磷酸化时，它会减少n端与Rho-GAP结构域的相互作用，从而使dcl1处于开放的活性构象。AKT是另一个直接磷酸化dcl1的激酶，位于Rho-GAP结构域n端的3条丝氨酸上。AKT可拮抗CDK5的作用，因为AKT磷酸化可使dcl1从开放的活性结构变为封闭的非活性结构，从而减弱dcl1的Rho-GAP和肿瘤抑制活性。AKT位于受体酪氨酸激酶（RTK）的下游，我们的数据表明，RTK配体刺激细胞后Rho-GTP的增加可归因于AKT的激活和DLC1的衰减。我们还发现SRC家族激酶（SFKs）是两种酪氨酸磷酸化dcl1的直接靶点，从而减弱dcl1的RHO-GAP和肿瘤抑制活性。一个酪氨酸位于RHO-GAP结构域，SFKs对其磷酸化会降低DLC1的RHO-GAP功能。另一种酪氨酸位于与张力蛋白结合所需的序列附近，其磷酸化可减少张力蛋白的结合。AKT和SFK的研究结果可能具有治疗潜力，因为与不表达DLC1的等基因肿瘤相比，AKT和/或SFK抑制剂治疗肿瘤异种移植物对表达DLC1的肿瘤具有更大的抗肿瘤活性。AKT和/或SFK抑制的高抗肿瘤活性与AKT和/或SFK对dcl1磷酸化的丧失以及dcl1的RHO-GAP和肿瘤抑制活性的重新激活有关。

Our growth regulation research has been concerned with oncogenes as positive regulators and tumor suppressor genes as negative regulators of normal and neoplastic growth. The main current project is focused on the molecular biology of the tumor suppressor gene DLC1, including the targets that regulate it and the targets that it regulates. DLC1 is inactivated frequently in a wide range of tumors, but many aspects of its mechanism of action remain incompletely understood. DLC1 negatively regulates Rho, via its Rho-GAP activity, and also encodes other activities. A major hypothesis is that DLC1 is frequently inactivated in cancer because it encodes a biologically important multifunctional protein. In support of this possibility, we have previously determined that DLC1 interacts with: 1) the tensin proteins, via an N-terminal region of DLC1 for which no function had been previously identified; 2) focal adhesion kinase (FAK) and talin, via a shared 8 amino acid motif; and 3) caveolin-1 (CAV-1), via the StAR-related lipid transfer (START) domain near the C-terminus of DLC1. DLC1 mutant analysis indicated that the binding sites for each of these interactions contributed to the growth suppressor function of DLC1, but that reduced binding seen with the mutants did not affect in vivo Rho-GAP activity of DLC1. These studies validate the hypothesis that DLC1 is a multifunctional protein whose biological activity depends both on its Rho-GAP activity and its ability to bind a variety of signaling molecules. We have also evaluated 12 cancers type in TCGA for the presence of point mutations in DLC1 and 9 other Rho-GAPs. The results indicate that DLC1 is the Rho-GAP that is mutated most frequently, with 5-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated seven of nine mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Point mutations in DLC1 were scattered throughout the coding sequences of the gene, implying that important biological functions are present throughout the protein. These results indicate that point mutation in DLC1 is an important mechanism for its decreased function in cancer, in addition to gene deletion and epigenetic changes. We have characterized how post-translational modifications of DLC1, especially phosphorylations, may affect its activity. We first identified CDK5, a cytoplasmic kinase whose physiologic role promotes differentiation, as a major activator of DLC1, which occurs via CDK5 phosphorylation of 4 serines in DLC1. When these serines, which are located N-terminal to the RHO-GAP domain of DLC1, are not phosphorylated, the N-terminus binds to the Rho-GAP domain, which places DLC1 in a closed, inactive conformation. When the serines are phosphorylated, it decreases the interaction of the N-terminus with the Rho-GAP domain, which places DLC1 in an open, active conformation. AKT is another kinase that directly phosphorylates DLC1, on 3 serines located N-terminal to the Rho-GAP domain. The effects of AKT antagonize those of CDK5, in that the AKT phosphorylations attenuate the Rho-GAP and tumor suppressor activities of DLC1 by changing DLC1 from an open, active configuration to a closed, inactive configuration. AKT lies downstream from receptor tyrosine kinases (RTKs), and our data indicate that increased Rho-GTP following stimulation of cells with RTK ligands is attributable to the activation of AKT and its attenuation of DLC1. We also identified SRC family kinases (SFKs) as direct targets for phosphorylating DLC1 on two tyrosines, which attenuate the RHO-GAP and tumor suppressor activities of DLC1. One tyrosine is located in the RHO-GAP domain, and its phosphorylation by SFKs reduces the RHO-GAP function of DLC1. The other tyrosine is located near the sequence required for binding tensin, and its phosphorylation reduces tensin binding. The findings with AKT and SFKs may have therapeutic potential, as treatment of tumor xenografts with AKT and/or SFK inhibitors has much greater antitumor activity against tumors that express DLC1 compared with isogenic tumors that do not express DLC1. The high antitumor activity from AKT and/or SFK inhibition is associated with loss of phosphorylation of the DLC1 by AKT and/or SFK together with reactivation of the RHO-GAP and tumor suppressor activities of DLC1.